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methylene blue milk

methylene blue milk - Microbiology Forum

methylene blue milk - Discuss Microbiology Science and Protocols here. Post questions on the study of viruses, fungi, parasites and bacteria here. Microbiology Forum.


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  #1  
Old 08-12-2006, 12:26 PM
vincent setiawan
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Default methylene blue milk



hai
i just want to know what's the formula for making methylene blue milk for detecting Streptococcus Group D and how to determine positive and negative reaction
thanks


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  #2  
Old 08-13-2006, 04:06 AM
JEDilworth
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Default methylene blue milk

Group D enterococci are identified in clinical micro labs by either a PYR
disc, or a combination of bile esculin agar with a 6.5% NaCl broth. I have
worked in micro for nearly 30 years and have never used or heard of
methylene blue milk. What type of laboratory (research? clinical?) are you
working in? I'm not saying that it doesn't work, it's just that I never
heard of it.

Group D enterococci are PYR +, BE+, 6.5% NaCl +

Group D nonenterococci are PYR -, BE+, 6.5% NaCl-

PYR can be bought commercially in disc form. You smear some of the colony on
it, wait two minutes, add reagent. If positive, it turns pink. Bile esculin
agar turns black if positive. Negative is no change from the yellowish agar
slant. The 6.5% NaCl broth is either cloudy from growth or clear from no
growth. You can also buy this broth with an indicator. It turns from
purplish blue to yellow if positive for growth.

PYR takes 2 minutes; the other two take overnight incubation.

Judy Dilworth, M.T. (ASCP)
Microbiology

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  #3  
Old 08-13-2006, 09:17 AM
N10
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Default methylene blue milk

HI

The MB test is an old method used to to assess quality of milk. It is a dye
reduction test where in a fixed aliqout of milk was incubated with a MB
solution. Reduction of the blue color or not within a fixed time gave a "
gun shot" indication of general microbial loading. It was usually
performed as part of a suite of tests with such media as the Croslley Milk
medium and peillicle assay for anaerobes and "bitty cream" formers.

Such tests were superseeded by more accurate specific and quatatative assays
in the Dairy Industry many years ago. I beleive ISO BS and FDA stndards
exist which describe current best practise for the examination of Diary
products.

Theoretically The MB test could be made relatively selective for Group D
strepps perhapes by inclusion of Kanamyciene and Aesculin in the
formualtion and with incubation at 44'c but its hardly worth it as so many
more suitable microbiological media are avialableof the shelf these day.

Good luck

N10





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  #4  
Old 08-14-2006, 04:52 AM
JEDilworth
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Default methylene blue milk

Thanks for the information. I knew someone out there would have some
information.

Yes, microbiology has come a long way even in the thirty years I have been
in the field. I have no idea what type of testing existed before my time.

Judy Dilworth, M.T. (ASCP)
Microbiology

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  #5  
Old 08-14-2006, 08:13 AM
N10
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Default methylene blue milk

HI

The Crossley Milk medium assay was perhapes the most elegant test of that
bygone era.

It permitted accurate detection and differentiation of three types of
Clostridial contamination in milk based on protein utilisation/degradation
and gas formation.

It was also duck egg blue which is my favourite colour

I wonder what other ancient microbiological techniques people know of ? or
still use ?

I remeber once just a few years back when we ran out of CO2 for the
genereation of anaerobic conditions I reverted to candling ! and it worked
very staisfactorily much to the amazement of the younger microbiologists who
witnessed it.

Best N10


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  #6  
Old 08-14-2006, 03:09 PM
JEDilworth
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Default methylene blue milk

I worked in a very small micro lab in the 70's and they were using a big
pickle jar and a candle for their chocolate and Thayer Martin plates. It
worked quite well. It was messy though, and somewhat tricky to position the
candle and light it so you wouldn't burn yourself.

I doubt whether it would be allowed in our more safety conscious
laboratories now.

I believe you meant 5% CO2 conditions, not anaerobic conditions. You need
gas generators for the latter.

Judy Dilworth, M.T. (ASCP)

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  #7  
Old 08-14-2006, 11:27 PM
Tony Stott
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Default methylene blue milk


JEDilworth wrote:

No, you need a brass anaerobic jar, a small metal mesh bag of palladium
catalyst, a water powered vacuum pump and basket ball bladder filled
from a cylinder of hydrogen, oh yes, don't forget to put your plates in
agar side down other wise the agar may fall into the lid when you apply
the vaccuum.

Anyone still know how to pull pasteur pipettes?

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  #8  
Old 08-15-2006, 03:14 AM
John Gentile
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Default methylene blue milk

On 2006-08-14 19:27:04 -0400, "Tony Stott" <[Only registered users see links. ]> said:


Do they even teach glass arts in micro any more? I used to make some
pretty good pipettes, and also glass "rakes" - a rod with a glass
trigangle at the end to rake up an entire plate of growth. I can still
teach the "kids" how to make wire loops, but they've never used them
with a bunsen burner!

BTW, a neat alternative for a decreased O2 environment is to use a
small jar that can fit plates and include a blood plate with a lawn of
E. coli. The E. coli can reduce the O2 to about the equivalent of a
candle jar. We used to make our own candles out of pathology paraffin.
--
John Gentile, MS M(ASCP)
Laboratory Information Manager
Providence, VAMC

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